Signaling Complexes in Renal Epithelial Cells

肾上皮细胞中的信号复合物

• 批准号: 6640447
• 负责人: Richard Tyler Miller
• 金额: $ 21.93万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6640447
• 关键词: G protein; JUN kinase; actin binding protein; biological signal transduction; calcium binding protein; calcium flux; caveolins; cell cell interaction; cell line; electron microscopy; enzyme induction /repression; epithelium; intracellular membranes; kidney cell; laboratory rat; mitogen activated protein kinase; parathyroid hormones; phospholipase C; protein localization; protein protein interaction; renal tubular transport; tissue /cell culture; transfection; yeast two hybrid system

DESCRIPTION (provided by applicant): The hypothesis guiding these studies is that the character and specificity of cell signaling systems is determined by structural proteins that organize multiple signaling proteins into discrete complexes in the cell. Some components are common to many signaling systems, while others may be unique. These studies will focus on the interaction of the extracellular Ca-sensing receptor (CaR) and filamin (ABP-280), a cytoskeletal protein, in renal epithelial cells with emphasis on MTAL and INCO cells. The CaR is a G protein-coupled receptor that responds to Ca and other polycations to regulate PTH secretion by the parathyroid glands and Ca, NaCI, KCl, and H20 transport in the kidney. The CaR has unique signaling properties that are not fully explained by its activation of Gi- and Gq-dependent signaling pathways. In an effort to identify potential scaffolding proteins for the signaling systems controlled by the CaR and identify new signaling pathways through which it might act, we used the CaR intracellular C-terminus as "bait" to screen a human kidney cDNA library using the yeast two hybrid approach. We found that the CaR interacts with filamin (ABP-280), a 280 kDa actin-binding cytoskeletal protein. Filamin also binds to variety of other signaling molecules including SEKI (MKK4), caveolin, the dopamine 02 receptor, Rho GTPases, SMADs and a- and Beta integrins, suggesting that it can act as a scaffold. The goals of this application are to define the interaction of the CaR and filamin and to understand how this interaction affects the signaling functions of the CaR in renal epithelial cells. The specific aims are: Aim 1) Characterize the interaction of the C-terminus of the CaR with fliamin using the yeast two hybrid system and mammalian cells; Aim 2) Determine the functional significance of the CaR-filamin interaction by analysis of CaR-stimulated signals in cells that lack filamin and by specific disruption of CaR-filamin association; Aim 3) Establish the subcellular pattern of distribution of the CaR in renal epithelial cells and determine if the interaction of the CaR with filamin contributes to the localization of the CaR in renal epithelial cells; Aim 4) Identify the smallest portion of filamin that can reconstitute signaling by the CaR in filamin-null cells, and then identify additional proteins that interact with that fragment. These studies will test the hypothesis that the interaction of filamin and the CaR is important for appropriate cell signaling by the CaR and that filamin acts as a scaffolding protein to organize the signaling pathway(s) regulated by the CaR.

描述（由申请人提供）：指导这些研究的假设是，细胞信号传导系统的特征和特异性是由结构蛋白决定的，结构蛋白将细胞中的多种信号传导蛋白组织成离散的复合物。一些组件对于许多信令系统是通用的，而另一些组件可能是独特的。这些研究将集中在细胞外钙敏感受体（CaR）和细丝蛋白（ABP-280），细胞骨架蛋白，在肾上皮细胞的MTAL和INCO细胞的重点相互作用。CaR是G蛋白偶联受体，其响应Ca和其他聚阳离子以调节甲状旁腺的PTH分泌以及肾脏中的Ca、NaCl、KCl和H2O转运。CaR具有独特的信号传导特性，其不能通过Gi和Gq依赖性信号传导途径的激活来完全解释。在努力确定潜在的支架蛋白的信号系统控制的CaR，并确定新的信号通路，通过它可能会采取行动，我们使用的CaR细胞内的C-末端作为“诱饵”，筛选人肾cDNA文库使用酵母双杂交的方法。我们发现，CaR与细丝蛋白（ABP-280），一个280 kDa的肌动蛋白结合细胞骨架蛋白相互作用。细丝蛋白还结合多种其他信号传导分子，包括SEKI（MKK 4）、小窝蛋白、多巴胺02受体、Rho GTP酶、SMAD以及α-和β整联蛋白，表明其可以充当支架。本申请的目的是定义CaR和细丝蛋白的相互作用，并了解这种相互作用如何影响肾上皮细胞中CaR的信号传导功能。具体目标是：目的1）利用酵母双杂交系统和哺乳动物细胞研究CaR C端与细丝蛋白的相互作用，目的2）通过分析缺乏细丝蛋白的细胞中CaR刺激信号和特异性破坏CaR与细丝蛋白的结合，确定CaR与细丝蛋白相互作用的功能意义;目的3）建立CaR在肾上皮细胞内的亚细胞分布模式，并探讨CaR与细丝蛋白的相互作用是否有助于CaR在肾上皮细胞内的定位;目的4）鉴定细丝蛋白的最小部分，其可以在细丝蛋白缺失的细胞中通过CaR重建信号传导，然后鉴定与该片段相互作用的其他蛋白质。这些研究将检验以下假设：细丝蛋白和CaR的相互作用对于CaR的适当细胞信号传导是重要的，细丝蛋白充当支架蛋白以组织由CaR调节的信号传导途径。